Several classes of small molecules that interact with double-stranded DNA have been identified. Many of these small molecules have profound biological effects. For example, many aminoacridines and polycyclic hydrocarbons bind DNA and are mutagenic, teratogenic, or carcinogenic. Other small molecules that bind DNA include: biological metabolites, some of which have applications as antibiotics and antitumor agents including actinomycin D, echinomycin, distamycin, and calicheamicin; planar dyes, such as ethidium and acridine orange; and molecules that contain heavy metals, such as cisplatin, a potent antitumor drug.
Most known DNA-binding molecules do not have a known sequence binding preference. However, there are a few small DNA-binding molecules that preferentially recognize specific nucleotide sequences, for example: echinomycin preferentially binds the sequence [(A/T)CGT]/[ACG(A/T)] (Gilbert et al.); cisplatin covalently cross-links a platinum molecule between the N7 atoms of two adjacent deoxyguanosines (Sherman et al.); and calicheamicin preferentially binds and cleaves the sequence TCCT/AGGA (Zein et al.).
The biological response elicited by most therapeutic DNA-binding molecules is toxicity, specific only in that these molecules may preferentially affect cells that are more actively replicating or transcribing DNA than other cells. Targeting specific sites may significantly decrease toxicity simply by reducing the number of potential binding sites in the DNA. As specificity for longer sequences is acquired, the nonspecific toxic effects due to DNA-binding may decrease. Many therapeutic DNA-binding molecules initially identified based on their therapeutic activity in a biological screen have been later determined to bind DNA.
Experiments performed in support of the present invention have identified an in vitro assay useful to screen for DNA-binding molecules. The assay also allows the discrimination of sequence binding preferences of such molecules. The potential therapeutic applications for molecules that bind to specific DNA sequences are widespread.